Virulence-Associated Enzymes of Cryptococcus neoformans

Fausto Almeida,a,b Julie M. Wolf,a Arturo Casadevalla,c

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USAa; Department of Cellular and MolecularBiology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazilb; Department of Molecular Microbiology and Immunology, Johns HopkinsBloomberg School of Public Health, Baltimore, Maryland, USAc

Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infec-tion process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better under-standing pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, tran-scriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This reviewfocuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C.neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalianhost. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology.

The facultative intracellular fungal pathogen Cryptococcus neo-formans is the causative agent of cryptococcosis, a disease that

primarily affects individuals with impaired immunity, such asthose with advanced HIV infection (1, 2). C. neoformans is a ubiq-uitous environmental fungus associated with both pigeon guanoand eucalyptus trees, and its environmental niche ranges from thetropical to the temperate (3). C. neoformans infection is acquiredfrom the environment via inhalation, after which it forms a localinfection in the lungs. This infection may be cleared, may be con-tained as a granuloma, or may disseminate from this initial site,leading to pneumonia and/or meningoencephalitis, the latter be-ing uniformly fatal if untreated. Despite the availability of antifun-gal therapy, more than 650,000 people die each year from C. neo-formans infection (1, 2, 4). The principal virulence factors of C.neoformans are a polysaccharide capsule, melanin production (5,6), the ability to grow at body temperature (7), and the secretion ofextracellular enzymes (7). These virulence factors confer a selec-tive advantage to C. neoformans for both residing in the environ-ment and in a mammalian host. Tightly controlled regulationleads to expression of enzymes required for fungal survival andhost damage once inside its mammalian host (8).

Many enzymes contribute to the composite cryptococcal viru-lence phenotype. Dissection of the pathogenic role of these en-zymes will enhance our understanding of cryptococcal pathogenicmechanisms and facilitate directed inhibitor development and/orvaccine discovery. We have included a table summarizing basicinformation regarding global C. neoformans enzymology (Table1) and a schematic displaying localization of most of the high-lighted enzymes discussed (Fig. 1). In this review, we discuss indetail the most important virulence-associated enzymes (Table 2),as well as additional target enzymes with potential for rationalantifungal drug design (Table 3). We examine this information inthe context of infection and analyze candidate target enzymes fordrug inhibition and vaccine discovery.

POLYSACCHARIDE CAPSULE

C. neoformans is the only fungal pathogen with a polysaccharidecapsule, an outermost polysaccharide structure located just out-side the cell wall. The two major polysaccharide capsule constitu-ents are glucuronoxylomannan (GXM) and glucuroxyloman-nogalactan (GXMGal) (9–11). GXM is the major component of C.

neoformans, a compound of �-1,3-linked mannose residues withxylosyl and glucuronyl side groups (12), whereas GXMGal is madeof �-1,6-linked galactose residues with xylose, mannose, andglucuronic acid (13). The capsule also contains nonpolysaccha-ride components, such as mannoprotein (MP) (10, 14, 15), al-though these MP components may represent transient compo-nents destined for cellular export.

The role of capsule in environmental growth is unknown, al-though speculations have been made that the capsule protects thefungus from desiccation or acts as a food source (16). Duringmammalian infection, the capsule participates in resisting phago-cytosis and modulating the immune response (17–21). Not onlyprotective against phagocytosis in both mammalian and lepi-dopteran hosts (22, 23), the capsule also protects the fungus afteringestion by serving as a free radical sink that can shield the cellfrom oxidative bursts (24). Hence, while the capsule is not part ofthe enzymatic microbial arsenal, the machinery responsible forcapsule synthesis and assembly does directly contribute to crypto-coccal virulence. The primary structures of GXM and GXMGalsubunits have been defined, but the mechanisms of subunit as-sembly into �106-Da branched structures have not (25, 26). Thedegree of branching and conformation of polysaccharides implyan elaborate assembly and regulatory enzymatic machinery (27).

The subunits of GXM and GXMGal are large glycans that re-quire several glycosyltransferases for synthesis. Both xylosyltrans-ferase and glucuronyltransferase activities are involved in capsularpolysaccharide biosynthesis (28–31). A xylosyltransferase, Cxt1,was the first glycosyltransferase identified with a defined role incapsule synthesis (31). It is a large transmembrane protein with�-1,2-xylosyltransferase activity (31), and deletion of the corre-sponding gene (CXT1) decreased capsular �-1,2-xylose linkagesand fungal growth in the lung in a mouse model of infection (30).

Several acapsular mutants were obtained through identifica-

Accepted manuscript posted online 9 October 2015

Citation Almeida F, Wolf JM, Casadevall A. 2015. Virulence-associated enzymes ofCryptococcus neoformans. Eukaryot Cell 14:1173–1185. doi:10.1128/EC.00103-15.

Address correspondence to Arturo Casadevall, arturo.casadevall@einstein.yu.edu.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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TABLE 1 Described enzymes in Cryptococcus neoformans

Enzyme Function(s)a EC no. Reference(s)

Localized on capsule and/or cell wall1,3-�-Glucan synthase Involved in �-glucan synthesis 2.4.1.34 135Acid phosphatase Involved in fungal cell adhesion to host tissues, localized in lysosomes,

and related to virulence (Table 2)3.1.3.2 106, 136, 137

Cas1 glycosyltransferase Participates in O-acetylation 2.4.1.X 138Chitin deacetylase Involved in chitin metabolism 3.5.1.41 139Chitin synthase Involved in chitin synthesis 2.4.1.16 140Chitinase Involved in chitin degradation 3.2.1.14 141Creatinine deaminase Involved in arginine and proline metabolism 3.5.4.21 142Esterase lipase Catalyzes hydrolysis of fatty acids 3.1.1.3 136GDP-mannose pyrophosphorylase Involved in GDP-mannose synthesis 2.7.7.13 143Glucan 1,3-�-glucosidase Involved in glucan synthesis 3.2.1.58 16Glucan 1,4-�-glucosidase Involved in glucan synthesis 3.2.1.3 16Gmt1 GDP-mannose Transport of GDP-mannose 2.7.7.22 144Lactonohydrolase Deficient strains show larger capsule size and facilitated immune

evasion3.1.1.15 37

N-Acetylgalactosaminoglycan deacetylase Involved in polysaccharide metabolism 3.1.1.58 145Phosphoaminase Involved in amino acid synthesis 136Phosphomannomutase Involved in GDP-mannose synthesis 5.4.2.8 143Phosphomannose isomerase Involved in GDP-mannose synthesis 5.3.1.8 143Uph1 ATPase Required for vesicle acidification 146Uxs1 decarboxylase Converts UDP-glucuronic acid to UDP-xylose 147�-1,3-Glucanase Involved in glucan synthesis 3.2.1.59 16�-Amylase Hydrolyzes alpha bonds of several polysaccharides and involved in cell

wall building3.2.1.1 148

�-Glucosidase Breaks down disaccharides to glucose and starch and involved in cellwall building

3.2.1.20 136

�-Mannosidase Involved in cell building through mannose metabolism 3.2.1.24 136�-Mannosyltransferase Involved in polysaccharide metabolism 2.4.1.132 38, 149�-Endoglucanase Involved in cell wall formation 3.2.1.4 148�-Glucosidase Involved in cell wall formation 3.2.1.21 136�-Glucuronidase Involved in cell wall formation, catalyzing breakdown of complex

carbohydrates3.2.1.31 136

Secreted/releasedAcyltransferase Involved in food acquisition 3.1.1.3 92Alkaline phosphatase Involved in regulation of signaling cascades and several protein

structure and localized in endoplasmic reticulum3.1.3.1 150

Aspartyl protease Involved in food acquisition 3.4.23.X 111Cellulase Involved in polysaccharide degradation 3.2.1.4 151DNase DNA degradation and related to virulence (Table 2) 3.1.21.1 79Metalloprotease Catalyzes mechanism that involves a metal and related to virulence

(Table 2)3.4.24.77 113, 152

Phospholipase B Similar to phospholipase C function, degrades cell membranecomponents, supports fungal attachment to host cells, localized oncell wall, and related to virulence (Table 2)

3.1.1.5 91, 92

Phospholipase C Degrades cell membrane components, supports fungal attachment tohost cells, and related to virulence (Table 2)

3.1.4.11 93

Protease Performs proteolysis interfering with host defense response 3.4.21.53 107, 108S2P endopeptidase Performs proteolysis 3.4.24.85 153Serine peptidase Performs proteolysis, coordinating several physiological functions 3.4.21.X 152Superoxide dismutase Catalyzes dismutation of toxic superoxide, converting superoxide to

hydrogen peroxide and oxygen and related to virulence (Table 2)1.15.1.1 83-85

Localized intracellularly2-Methylcitrate synthase Converts acyl groups into alkyl groups on transfer 2.3.3.5 1543-�-Hydroxysteroid 3-dehydrogenase Oxidizes a substrate by reduction reaction that transfers 1 or more

hydrides to electron acceptor1.1.1.270 155

6-Phosphogluconate dehydrogenase Involved in production of ribulose 1.1.1.44 156, 157Acetate kinase Catalyzes formation of acetyl-CoA 2.7.2.1 158Aconitase Catalyzes isomerization of citrate to isocitrate and involved in

response to nitrosative stress4.2.1.3 159

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TABLE 1 (Continued)

Enzyme Function(s)a EC no. Reference(s)

Adenylyl cyclase Cac1 Converts ATP to cAMP 4.6.1.1 160Alternative oxidase Part of electron transport chain in mitochondria 1.10.3.11 161Aminopeptidase Catalyzes cleavage of amino acids from amino terminus of protein 3.4.11.21 137C-9-methyltransferase Involved in glycosphingolipid pathway 2.1.1.129 127Can2 carbonic anhydrase Responds directly to intracellular carbon oxide 4.2.1.1 162, 163Casein kinase 1 Dephosphorylation of Hog1 under stress conditions 2.7.11.1 164Catalase Protects cells from oxidative damage by reactive oxygen species 1.11.1.6 137, 150Cytochrome c peroxidase Takes reduced equivalents from cytochrome c and reduces hydrogen

peroxide to water1.11.1.5 165

Deacetylase Removes acetyl groups from lysine in proteins and is localized in cellwall

3.5.1.108 166

Dolichyl-diphosphooligosaccharide-proteinglycotransferase

Participates in N-glycan biosynthesis 2.4.99.18 167

Ferrochelatase Catalyzes final step in heme biosynthesis from highly photoreactiveporphyrins

4.99.1.1 168

Flippase Participates in phospholipid translocation between membrane sidesand localized in cell wall

3.6.3.1 169, 170

Glucose-6-phosphate dehydrogenase Is in pentose phosphate pathway, maintaining the level of coenzymeNADPH

1.1.1.49 171

Glucose-phosphate isomerase Catalyzes conversion of glucose-6-phosphate into fructose6-phosphate

5.3.1.9 172

Glucosylceramide synthase Involved in glucosylceramide synthesis, localized in cell wall, andrelated to virulence (Table 2)

2.4.1.80 127, 128

Glucuronyltransferase Involved in biosynthetic pathway of O-acetylated mannan 2.4.1.17 28Glutathione peroxidase Protects cells from oxidative damage 1.11.1.9 173Glyoxal oxidase Copper metalloenzyme that catalyzes oxidation of aldehydes to

corresponding carboxylic acids coupled to reduction of dioxygen toH2O2

1.2.1.23 148

Homoisocitrate dehydrogenase Participates in lysine biosynthesis 1.1.1.87 115Homoserine kinase Participates in glycine, serine, and threonine metabolism 2.7.1.39 174Homoserine O-acetyltransferase Participates in methionine and sulfur metabolism 2.3.1.31 175Hyaluronic synthase Involved in production of glycosaminoglycan at cell surface 2.4.1.212 176Imidazole glycerol-phosphate dehydratase Participates in histidine biosynthesis 4.2.1.19 177IMP dehydrogenase Participates in GTP biosynthesis 1.1.1.205 178Inositol phosphotransferase 1 Involved in glycosphingolipid pathway 2.7.1.X 127Inositol-phosphorylceramide synthase Involved in glycosphingolipid pathway 2.7.1.X 179Ire1 kinase Involved in cellular response to unfolded proteins 2.7.11.1 180Isocitrate lyase Catalyzes cleavage of isocitrate to succinate and glyoxylate 4.1.3.1 181Laccase Polyphenol oxidase and copper-containing oxidase enzyme, localized

in cell wall, and related to virulence (Table 2)1.10.3.2 45, 46, 50

Malate dehydrogenase Catalyzes oxidation of malate to oxaloacetate 1.1.1.37 182Mannitol-1-phosphate 5-dehydrogenase Participates in fructose and mannose metabolism 1.1.1.17 183, 184Mannose-1-phosphate guanylyltransferase(GDP)

Participates in fructose and mannose metabolism 2.7.7.22 144

Mannosyl phosphorylinositol ceramidesynthase

Involved in glycosphingolipid pathway 2.4.X.X 127

Mannosyltransferase Participates in O-mannosylation of proteins and involved in cell wallintegrity and morphogenesis

2.4.1.109 185

Myristoyl-CoA: protein N-myristoyltransferase Catalyzes transfer of myristate from CoA to proteins 2.3.1.97 116Pde1 phosphodiesterase Modulates cAMP 3.1.4.1 186Phosphoglucomutase Participates in interconversion of glucose 1-phosphate and glucose

6-phosphate5.4.2.2 172

Protein farnesyltransferase Participates in formation of farnesyl protein and diphosphate 2.5.1.58 187Rho1 GTPase Involved in MAPK cascade 3.6.5.2 188RNase III Binds and cleaves double-stranded RNA 3.1.26.3 189Saccharopine dehydrogenase Participates in lysine metabolism 1.5.1.10 190Sphingolipid methyltransferase 1 Participates in methylation of glucosylceramide 2.1.1.1 191Sterol 14�-demethylase Involved in sterol metabolism 1.14.13.7 192Sterol 24-C-methyltransferase Involved in sterol metabolism 1.15.1.1 193Thiol peroxidase Reduces peroxides and inhibits hydrogen peroxide response 1.11.1.7 194

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tion of rough colonies. This type of screen identified four genesrequired for capsule formation: CAP10, CAP59, CAP60, andCAP64. Although these genes are not essential, their mutationdoes confer defects in growth and in mouse models of infection(17, 32–35). Cells from these mutant strains lacked or produced

extremely reduced capsule, but these mutations did not corre-late with enzymatic deficiency in UDP-glucose dehydrogenase,UDP-glucuronate decarboxylase, UDP-glucuronyl:acceptortransferase, UDP-xylosyl:acceptor transferase, or lipid-linkedoligosaccharide biosynthetic pathways. CAP10 is a putative xy-

TABLE 1 (Continued)

Enzyme Function(s)a EC no. Reference(s)

Thioredoxin reductase Catalyzes reduction of thioredoxin 1.8.1.9 195Threonine synthase Participates in glycine, serine, and threonine metabolism 4.2.3.1 174Thymidylate synthase Catalyzes conversion of dUMP to deoxythymidine monophosphate 2.1.1.45 196Transaldolase Involved in pentose phosphate pathway 2.2.1.2 159Trehalose-6-phosphate phosphatase Participates in starch and sucrose metabolism 3.1.3.12 197Trehalose-6-phosphate synthase Participates in starch and sucrose metabolism 2.4.1.15 197UDP-galactopyranose mutase Catalyzes conversion of UDP-D-galactopyranose in

UDP-D-galacto-1,4-furanose5.4.99.9 198

UDP-glucose dehydrogenase Participates in conversion of UDP-glucose to UDP-glucuronate, andformation of glycosaminoglycans

1.1.1.22 199

UDP-glucuronate decarboxylase Participates in nucleotide sugar metabolism 4.1.1.35 147Urease Catalyzes hydrolysis of urea into carbono dioxide and ammonia and

related to virulence (Table 2)3.5.1.5 74

Xylosylphosphotransferase Participates in O-glycosylation biosynthesis and related to virulence(Table 2)

2.7.8.32 28, 31, 200

�8 desaturase Involved in glycosphingolipid pathway 1.14.19.4 127a cAMP, cyclic AMP; MAPK, mitogen-activated protein kinase.

FIG 1 Enzymes are crucial for fungal pathogenesis and can alter the infection process. These enzymes are potential targets for new antifungal agents. (A) Somepathogenesis-related enzymes are retained to be active inside the cell body, while others are secreted. Some, like laccase, are both retained and secreted. (B) Ofthose released, some are secreted using traditional secretion systems, while others are included as cargo in extracellular vesicles.

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losyltransferase gene, and cap10� mutants show a pleiotropicphenotype, which includes enlarged cell size, smaller extracel-lular vesicles, and affected expression of some virulence factors(36). CAP10 therefore is required for both capsule formationand other aspects of fungal virulence.

Capsular lactonohydrolase also affects multiple capsule-re-lated phenotypes (37). A strain lacking lactonohydrolase (lhc1�)produced capsules with a larger size and altered branching, den-sity, and solvation compared to the parental strain. These capsularstructure alterations increased virulence in murine infection (37).Taken together, these results suggest that lactone may be involvedin cross-linking of the capsule.

�-1,3-Mannosyltransferase (encoded by CMT1) synthesizesthe mannose backbone of GXM and thus plays a crucial role incapsule synthesis. However, �-1,3-mannosyltransferase activity ismore involved in in serotype A capsule biosynthesis than in theserotype D C. neoformans (38, 39). Serotypes A and D representtwo of the four C. neoformans serotypes: C. neoformans var. neo-formans (serotypes A and D) and C. neoformans var. gattii (sero-types B and C), which can be distinguished according to theirgrowth differences on diagnostic media (40). The strain-specificcapsule synthesis differences, such as the role of CMT1, show theimportance of studying multiple strain backgrounds.

Much remains to be learned about the enzymatic machineryinvolved in capsule synthesis, including enzyme localization andkinetics. Detailed studies of capsule structure and the enzymaticmachinery involved are critical for a better understanding of thefunction of the capsule production and regulation.

MELANIN SYNTHESIS

Melanin formation protects C. neoformans from oxidative damageas well as from both heat and cold (41, 42). Melanin is synthesized

on 2,3- or 3,4-diphenol substrates by a phenoloxidase and accu-mulates in the C. neoformans cell wall (43, 44). The melanin-syn-thesizing enzyme has two classical laccase characteristics: a glyco-sylated copper-containing protein with the ability to oxidizediphenolic substrates and the ability to produce decarboxy dop-achrome (45, 46). C. neoformans melanin synthesis occurs only inthe presence of exogenous dihydroxyphenols, since no known C.neoformans endogenous substrate exists. Several diphenols canserve as the substrates for pigment synthesis by C. neoformanslaccase (47), such as the substrates consisting of para- and ortho-diphenols, monophenols, L-dopa, and esculin, indicating that theenzyme has broad specificity and the ability to generate pigmentsfrom different compounds (47–53). Iron increases laccase activ-ity, but hydrogen peroxide has no effect on enzymatic activity,despite the antioxidant properties of melanin (54).

The genes LAC1 and LAC2 encode two laccases, but a singledeletion in LAC1 is able to prevent melanin production (55–58).Lac1 localizes in the cell wall, while Lac2 is cytoplasmic, but Lac2can localize to the cell wall in the absence of Lac1 (55). lac1�mutants are easily identified as white colonies when cultivated oncatecholamine-containing media (59). The lac1� mutant showsdecreased virulence in survival studies with rabbit infection (59),corroborating the important role in the fungal virulence (5, 46). Inaddition to its cell wall localization, laccase is packaged into extra-cellular vesicles, a nontraditional mechanism of secretion, and cantherefore mediate damage away from the laccase-producing fun-gal cell (Fig. 1).

Melanin is considered a powerful antioxidant, since it mayprotect cryptococcal cells against oxygen- and nitrogen-derivedoxidants of the type made by host effector cells (5, 60–62). Inaddition to its capacity to absorb free radical fluxes, melanin canalso contribute to acquired resistance against to the antifungals

TABLE 2 Enzymes related to the virulence in Cryptococcus neoformans

Enzyme Comment(s) Reference(s)

Acid phosphatase Deficient strains show affected virulence in mouse and Galleria mellonella models of infection 106DNase Acts in degrading host DNA and supplies C. neoformans with nucleotides 79Glucosylceramide synthase Required for virulence in murine model of infection 127, 128Laccase Deficient strains show decreased virulence in survival studies with rabbit and mouse models of infection 59Mannosyltransferase Required for virulence in murine model of infection 185Metalloprotease Deficient strains unable to cross endothelium in in vitro model of human blood-brain barrier and is

required for invasion of central nervous system113

Phospholipase B Required in invasion of host tissue and dissemination in murine model 95Phospholipase C Shown to be important for several virulence phenotypes 101, 102Superoxide dismutase Attenuated growth of deficient strains within macrophages 89Urease Deficient strains less virulent than wild-type strain in mouse model of infection and is involved in

fungal escape from lung to cross blood-brain barrier76

Xylosylphosphotransferase Deficient strains manifest reduced growth in lung tissue in mouse model of infection 30

TABLE 3 Possible target enzymes for rational antifungal drug design

Enzyme(s) Comment(s) Reference(s)

14�-Demethylase A critical enzyme in sterol assembly 119Glucosylceramide synthase Glucosylceramide plays critical role in pathogenicity of C. neoformans 127, 128Laccase Melanization aids virulence 60, 63, 64, 65Myristoyltransferase Myristoylation inhibition is fatal for C. neoformans 116, 117Phosphoribosylaminoimidazole carboxylase Mutants that cannot synthesize adenine have reduced virulence 114Pyrophosphorylase and cytosine-specific permease Enzymes are basis of C. neoformans flucytosine resistance 201, 202Sterol synthesis enzymes Sterol synthesis enzyme mutants show resistance to fluconazole and amphotericin 122-124

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amphotericin B and caspofungin, since nonmelanized cryptococ-cal cells are more susceptible than melanized cells to amphotericinB and caspofugin. Moreover, killing assays demonstrated that ad-dition of melanin particles to amphotericin B or caspofungin sig-nificantly reduces their toxicities against C. neoformans (63–65).Thus, melanin and laccase are considered promising targets fordrugs against C. neoformans infection.

EXTRACELLULAR ENZYMES

As nature’s “recyclers,” environmental fungi secrete a number ofdegradative enzymes to breakdown macromolecules and obtainnutrients in the environment (7, 66–69). C. neoformans is no ex-ception and releases a number of lipases, proteases, and DNases.However, during the infection process, the same degradative en-zymes contribute to virulence by destroying tissues, promotingfungal survival, and interfering with effective immune responses.

Urease is almost universally expressed by C. neoformans iso-lates. In the environment, C. neoformans is often isolated fromavian excreta (70, 71). To survive and grow on this medium, thefungus must metabolize creatinine, xanthines, and uric acid. Highurease activity may benefit the fungus under these conditions (72–74), as the enzyme catalyzes the hydrolysis of urea to ammoniaand carbamate. Urease is considered a major cryptococcal viru-lence factor (75). A urease knockout (URE1) strain of C. neofor-mans was significantly less virulent than the wild-type strain in amouse model of infection (76). Urease plays a role in fungal escapefrom the lung to cross the blood-brain barrier but is not requiredfor fungal growth once inside the brain (76). Urease productionvaries among clinical isolates; however, the vast majority (99.6%)demonstrate some level of urease activity (74, 77, 78). Neverthe-less, occasional urease-negative variants have been isolated in clin-ical isolates (77), suggesting that this enzyme can be dispensable,provided that there are compensatory virulence mechanisms.

Extracellular DNase is produced by C. neoformans in highquantities (79). This DNase may degrade host DNA secreted byneutrophils as part of the innate immune response (80) and addi-tionally may supply C. neoformans with nucleotides. A survey ofseveral yeast species, including C. neoformans, suggests a correla-tion between urease activity and extracellular DNase production(79). DNase activity is stronger in clinical strains than in environ-mental strains, further suggesting DNase may play a role as a vir-ulence factor (81).

Superoxide dismutases (SODs) convert superoxide to hydro-gen peroxide and oxygen (82). Two SODs have been described inC. neoformans (83–88). SOD contributes to virulence of C. neofor-mans by facilitating growth within macrophages (89), through amechanism that is likely to involve protection of the fungusagainst superoxide generated by host immune response (2). In thisregard, melanin and SOD may stimulate complementary defensesfor the C. neoformans cells’ protection against oxidative damage.SOD production is regulated by temperature, with increases inexpression at 37°C compared to 25°C. Thus, increased SOD pro-duction at body temperatures may protect the fungus against ox-idizing agents produced from host effector cells (90).

Phospholipases degrade cell membrane phospholipids in anenzyme-dependent mechanism. C. neoformans extracellular su-pernatants contain phospholipase B, phospholipase C, lysophos-pholipase, and acyltransferase (91–93), and phospholipase activ-ity supports fungal attachment to host cells (94). Phospholipase Bpromotes fungal invasion of host tissue (95) and hydrolyzes phos-

pholipids in lung surfactant and the plasma membrane (92, 96).Moreover, it contributes to fungal survival by maintaining cellwall integrity (97) and provides nutrients that can be used as solecarbon sources by C. neoformans during the infection (98, 99). Asdescribed above, it has also been localized to the cell wall (97), andits transport to the plasma membrane and cell wall is N-glycandependent (100). Phospholipase C is crucial for several virulencephenotypes (melanin production, growth at 37°C, phospholipaseB secretion, and antifungal drug resistance) and is also involved inhomeostasis regulation, cell separation following cytokinesis, andcell wall integrity (101, 102).

Phosphatases remove a phosphate group from their substratesand play important roles in regulating protein structure and sig-naling cascades (103, 104). A secreted acid phosphatase is involvedin fungal cell adhesion to host tissues, suggesting an importantrole in establishing infection (105). Acid phosphatase is encodedby the gene APH1 in C. neoformans. In both wax worm and mu-rine models of cryptococcosis, aph1� strain-infected animals sur-vived longer than those in the wild-type-infected model (106),demonstrating the importance of this enzyme during infection.

Proteases break down proteins and are considered importantvirulence factors, contributing to tissue invasion, colonization,and alteration of the host defense response. Protease activity in C.neoformans cultures has been reported by several investigators(107–111). Proteases play important roles in host cell penetrationand virulence of C. neoformans (112). Recently, a metalloproteasewas identified by proteomic analyses of the extracellular proteinsfrom C. neoformans and found to be required for invasion of thecentral nervous system in murine infection of C. neoformans(113). Moreover, the metalloprotease knockout (mpr1�) strainwas unable to cross the endothelium in an in vitro model of thehuman blood-brain barrier (113).

DRUG DESIGN AND RESISTANCE

Definition of enzymatic pathways can provide crucial targets forantimicrobial drug design. One way to identify targets is to iden-tify unique metabolic requirements for cryptococcal growthand/or virulence. An example of this is the C. neoformans phos-phoribosylaminoimidazole carboxylase gene (ADE2). Mutantswith mutations in this gene lack an enzyme required for adeninesynthesis and thus have reduced virulence compared to the wild-type strain (114). This observation suggests potential for rationaldrug design utilizing differences in adenine synthesis pathwaysbetween host and pathogen (as first suggested in reference 7).Several candidate enzymes in C. neoformans have been studiedregarding fungal amino acid synthesis (e.g., homocitrate synthase,homoisocitrate dehydrogenase, �-aminoadipate reductase, sac-charopine reductase, and saccharopine dehydrogenase) (115).However, comparisons between C. neoformans var. neoformansand C. neoformans var. gattii have shown that candidate targets donot necessarily translate across Cryptococcus species. Saccharopinereductase, an enzyme involved in lysine synthesis, was not de-tected in C. neoformans var. gattii but was detected in C. neofor-mans var. neoformans. This C. neoformans var. gattii strain wasable to grow even in the absence of lysine (115), indicating thatfurther research to identify enzymes essential across all Cryptococ-cus species is required.

Another essential process for C. neoformans is protein myris-toylation. C. neoformans myristoyltransferase catalyzes the trans-fer of myristate from coenzyme A (CoA) to the amino-terminal

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glycine residue of a subset of cellular proteins, and this enzyme isessential for C. neoformans viability (116, 117). N-Myristoyl pro-teins and myristoylation inhibition by the myristic acid analog4-oxatetradecanoic acid are crucial for this organism (118). Thus,therapies directed at myristoylation may also be a possible targetfor rational antifungal drug design.

In some cases, an antifungal target is well defined, but multipleenzymes involved in target synthesis provide several inhibitorystrategies. Sterols and their synthetic pathways are major antifun-gal targets in many fungi, but resistance leads to difficulties inpatient treatment. Fluconazole-resistant strains require a 10-fold-higher drug concentration to inhibit sterol 14�-demethylation(119), rendering the drug clinically unfeasible. The molecular ba-sis for differential enzyme function has been identified in severalclinical C. neoformans strains (120). One documented flucona-zole- and amphotericin-resistant C. neoformans patient isolateshowed reduced relative sterol content and a defect in �-8-isomer-ase, depleted ergosterol, and accumulated aberrant �-8-double-bonded ergosterol precursors (121, 122), suggesting the ability toform membrane pores due to aggregation and formation of am-photericin-ergosterol complexes. Another study evaluating flu-conazole- and amphotericin-resistant isolates observed reducedergosterol content in the isolates, as well as reduced sensitivity ofP450 14�-demethylase to inhibition by fluconazole, and a defectin sterol �8-�7 isomerase (123). Another C. neoformans strainwith defective sterol �8-�7 isomerase was discovered in an am-photericin B-resistant isolate from an AIDS patient (124). Thesemutations in sterol synthesis enzymes explain resistance evolutionand generate targets to fight it with. This information can also helpin rational drug design methodologies.

Identification of key virulence-related enzymes is yet anotherroute toward finding an effective drug target. Glycosphingolipidsare essential to regulate survival and/or replication of C. neofor-mans in the phagolysosome, as well as in the extracellular environ-ment of the host (125–127). Glucosylceramide plays critical role inpathogenicity of C. neoformans, since glucosylceramide synthase(Gcs1) is required for virulence in the murine model of infection(128). gcs1� mutants corroborate the crucial role of the glycosph-ingolipid synthesis in regulation of this considerable aspect of C.neoformans virulence (127). Thus, the glycosphingolipid pathwaymay also be a reasonable target for antifungal therapies.

Laccase has been considered a drug target in C. neoformansbecause melanization is critical to virulence. Inhibition of fungalmelanization in murine infection using the herbicide glyphosateprolonged average mouse survival. Glyphosate is an inhibitor ofboth the shikimate acid pathway and L-dopa polymerization(129). Thus, therapies directed at melanization may also be a po-tential target for antifungal drug design.

Occasionally, a drug proven to work on one microbial patho-gen will also be effective against another. This appears to be thecase with several viral medications. Drugs such as indinavir andoseltamivir inhibit human immunodeficiency virus (HIV) pro-tease or influenza virus neuraminidase, respectively, and demon-strate the impact an enzymatic inhibitor can have in the clinic(130, 131). The use of protease inhibitors has shown positive ef-fects on C. neoformans and Candida albicans infections, wheredrug treatment was associated with inhibition of fungal growthand proliferation in vitro (132, 133). These are likely inhibiting thefungal proteases, both cell associated and as part of the fungalsecretome.

CONCLUSION

Recent advances in genomics, proteomics, transcriptomics, andmass spectrometry have facilitated the identification and charac-terization of new fungal enzymes, including those specific to bothfungi and C. neoformans. These enzymes are required for manyimportant biological processes, including growth and infection.The importance of the secretome in cryptococcal pathogenesis isapparent from the fact that strain differences in secreted enzymescorrelate with their virulence (134). Nonetheless, important ques-tions remain. Future research on cryptococcal enzymology willnot only identify new enzymes and their roles during infection butalso pinpoint enzymatic targets for the development of antifungalagents.

ADDENDUM IN PROOF

There are, of course, many enzymes involved in signaling cas-cades, most of which were not discussed in this review. One suchenzyme is vital to stress response in C. neoformans and otherpathogenic fungi and thus merits a well-deserved mention: thecalcium-dependent phosphatase calcineurin (W. J. Steinbach, J. L.Reedy, R. A. Cramer, Jr., J. R. Perfect, J. Heitman, Nat Rev Micro-biol 5:418 – 430, 2008). This enzyme is required for growth in amammalian host and therefore is necessary to cause disease (A.Odom, S. Muir, E. Lim, D. L. Toffaletti, J. Perfect, J. Heitman,EMBO J 16:2576 –2589, 1997). Studies utilizing calcineurin inhib-itors for invasive disease in animal models have shown promisingresults, and this work is now moving into translational stages(D. P. Kontoyiannis, R. E. Lewis, B. D. Alexander, O. Lortholary,F. Dromer, K. L. Gupta, G. T. John, R. del Busto, G. B. Klintmalm,J. Somani, G. M. Lyon, K. Pursell, V. Stosor, P. Munoz, A. P.Limaye, A. C. Kalil, T. L. Pruett, J. Garcia-Diaz, A. Humar, S.Houston, A. A. House, D. Wray, S. Orloff, L. A. Dowdy, R. A.Fisher, J. Heitman, N. D. Albert, M. M. Wagener, N. Singh, Anti-microb Agents Chemother 52:735–738, 2008, http://dx.doi.org/10.1128/AAC.00990-07). Other enzymes involved in stress re-sponses may similarly be identified and targeted in the future.

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